Chapter 5

Molecular Tools for

Studying Genes and
Gene Activity

Course Objectives:
 Methods used to study structure and function of genes.
 Molecular separation using gel electrophoresis for both nucleic
acids and proteins.
 Use of labeled tracers to detect tiny amounts of nucleic acids and
proteins.
 Assaying DNA-protein and protein-protein interactions.
 Student Expected Learning Outcomes
1. Discuss experiments used by molecular biologists to
separate nucleic acids and proteins.
2. Explain how gel electrophoresis, ion exchange
chromatography, and gel filtration can be used to
separate nucleic acids and proteins.
3. Understand and describe how labeled tracers are
used to detect small amounts of DNA, RNA and
proteins.
4. Explain the various assays used to detect protein-
DNA and protein-protein interactions, and know
when to apply them.

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 5.1 Molecular Separations
 Often mixtures of proteins or nucleic
acids are generated during the course
of molecular biological procedures.
A protein or a nucleic acid may need to be
purified from a crude cellular extract
 Gel electrophoresis is used to separate
different species of:
Nucleic acids
Proteins

5-3
 DNA Gel Electrophoresis

 Melted agarose is poured

into a tray equipped with
a removable comb.
 Comb “teeth” form slots
in the solidified agarose.
 DNA samples are placed
in the slots.
 An electric current is
run through the gel at a
neutral pH to allow the
sample to travel through
the gel matrix.
5-4
 DNA Separation by Agarose Gel
Electrophoresis
 DNA is negatively charged due to the
phosphates in its backbone and moves
toward the positive pole
 Small DNA pieces have little
frictional drag so they move rapidly
 Large DNAs have more frictional drag
so their mobility is slower
 Distributes DNA according to size
• Largest near the top
• Smallest near the bottom
 DNA is stained with fluorescent dye
(ethidium bromide) that intercalates
between the bases
5-5
 Electrophoresis of Large DNA
 Special techniques are
required for DNA fragments
larger than about 0.1 Mb.
 Instead of constant current,
alternate long pulses of current
in forward direction with
shorter pulses in either
opposite or sideways direction.
 Technique is called pulsed-field
gel electrophoresis (PFGE),
used to separate yeast
chromosomes (0.2-2.2Mb)
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 Protein Gel Electrophoresis
 Separation of proteins is done using
polyacrylamide gel electrophoresis
(PAGE).
Treat proteins to denature subunits with
detergent such as sodium dodecyl sulfate
(SDS)
• SDS coats polypeptides with negative charges so
all move to anode (+ electrode)
• Masks natural charges of protein subunits so all
move relative to mass not charge
As with DNA, smaller proteins move faster
toward the anode
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 Protein Gel Electrophoresis Cont.

 Protein can be separated by SDS-PAGE and detected by Western blotting

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 Summary
 DNAs, RNAs, and proteins of various
masses can be separated by gel
electrophoresis.
 Most common gel used in nucleic acid
electrophoresis is agarose, but
polyacrylamide is typically used in
protein electrophoresis.
 SDS-PAGE is used to separate
polypeptides according to their masses.

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 Ion-Exchange Chromatography
Ion-exchange chromatography uses a resin to
separate substances by charge (e.g. DEAE+,
Phosphocellulose/P11-).
 This is especially useful for proteins, and
uses buffer containing negative ions/cation
(DEAE+) or positive ions/anion (P11-) to
compete with proteins.
 Resin is placed in a column and the sample is
loaded onto the column material.

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 Separation by Ion-Exchange
Chromatography
 Once the sample is
loaded, buffer is
passed over the resin +
sample.
 As ionic strength of
elution buffer
increases, samples of
solution flowing
through the column are
collected.
 Samples are tested
for the presence of the
protein of interest.
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 Gel Filtration Chromatography
 Protein size is a valuable property that can
be used as a basis of physical separation.
 Gel filtration uses columns filled with porous
resins that let in smaller substances and
exclude larger substances.
 As a result larger substances travel faster
through the column.

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 Affinity Chromatography
 The resin contains a substance to which
the molecule of interest has a strong and
specific affinity.
 The molecule binds to a column resin
coupled to the affinity reagent (e.g.
Chapter 4  Oligohistidine tagged protein-Nickel column).
 Molecule of interest is retained
 Most other molecules flow through
 Last, the molecule of interest is eluted from
the column using a specific solution that
disrupts their specific binding

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 Summary

 Ion-exchange chromatography can be used to

separate substances according to their size
and charge.
 Gel filtration chromatography uses columns
filled with porous resins that let in smaller
substances but exclude larger ones.
 Affinity chromatography is a powerful
purification technique that exploits an affinity
reagent with strong and specific affinity for a
molecule of interest (His-tag, Flag-tag, GST-
tag, Myc-tag, HA-tag).
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 5.2 Labeled Tracers
• For many years “labeled” has been
synonymous with “radioactive”.
• Radioactive tracers allow vanishingly
small quantities of substances to be
detected.
• Molecular biology experiments typically
require detection of extremely small
amounts of a particular substance.

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 Autoradiography
 Autoradiography is a means
of detecting radioactive
compounds with a
photographic emulsion.
 Preferred emulsion is x-ray
film
 DNA is separated on a gel and
radiolabeled
 Gel is placed in contact with x-
ray film for hours or days
 Radioactive emissions from the
labeled DNA expose the film
 Developed film shows dark
bands
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 Autoradiography Analysis

 Relative quantity of
radioactivity can be assessed
looking at the developed film.
 More precise measurements
are made using a
densitometer.
 Area under peaks on a tracing
by a scanner
 Proportional to darkness of the
bands on autoradiogram

5-17
 Liquid Scintillation Counting
 Radioactive emissions from a
sample create photons of visible
light that are detected by a
photomultiplier tube in the
process of liquid scintillation
counting.
 Remove the radioactive material
(band from gel) to a vial containing
scintillation fluid
 Fluid contains a fluor that fluoresces
when hit with radioactive emissions
 Acts to convert invisible
radioactivity into visible light
 Bursts of light are recorded as
counts per minute (cpm)
5-18
5.3 Using Nucleic Acid Hybridization

 Hybridization is the ability of one

single-stranded nucleic acid to form a
double helix with another single strand
of complementary base sequence.
 Previous discussion focused on colony
and plaque hybridization.
 This section looks at techniques for
isolated nucleic acids.

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 Southern Blots: Identifying
Specific DNA Fragments
 Digests of genomic DNA are separated on a gel.
 The separated pieces are transferred to filter
(nitrocellulose) by diffusion, or more recently by
electrophoresing the DNA onto the filter.
 The filter is then treated with alkali to denature
the DNA, resulting ssDNA binds to the filter.
 A labeled cDNA probe that is complementary to
the DNA of interest is then applied to the filter.
 A positive band should be detectable where
hybridization between the probe and DNA occurred.

5-20
 Southern Blot
 The probe hybridizes and
a band is generated
corresponding to the DNA
fragment of interest.
 Visualize bands with x-
ray film or autoradio-
graphy.
 Multiple bands can lead
to several interpretations.
 Multiple genes
 Several restriction sites in
the gene
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 Summary
 Labeled DNA (or RNA) probes can be used to
hybridize to DNAs of the same or very similar
sequence on a Southern blot (Northern blot).
Autoradiography can be used to measure
quantities of nucleic acids present, and so do
Phosphorimaging and scintillation liquid
counting.
 DNA southern blot can be used to identify
target genes and determine copy number, but
it is not accurate as real time PCR .

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 5.4 DNA Sequencing
 Sanger, Maxam, and Gilbert developed 2
methods for determining the exact base
sequence of a cloned piece of DNA.
 Modern DNA sequencing is based on the Sanger
method and uses dideoxy nucleotides to
terminate DNA synthesis.
 The process yields a series of DNA fragments whose
size is measured by electrophoresis
 The last base in each fragment is known as that
dideoxy nucleotide that was used to terminate the
reaction
 Ordering the fragments by size tells the base
sequence of the DNA
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Sanger Method of DNA Sequencing

5-24
 Automated DNA Sequencing

 Manual sequencing is
powerful but slow.
 Automated sequencing uses
dideoxynucleotides tagged
with different fluorescent
molecules.
Products of each
dideoxynucleotide will
fluoresce a different color
Four reactions are completed,
then mixed together and run
out on one lane of a gel
5-25
 Restriction Map Example
 Consider a 1.6 kb piece of
DNA as an example.
 Cut sample of the original
1.6 kb fragment with a
different restriction
enzyme.
 Separate the digests on an
agarose gel to determine
the size of pieces from
each digest.
 Can also use same digest to
find the orientation of an
insert cloned into a vector.
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 Summary
• DNA sequencing can allow us to determine the
exact sequence of DNA being studied.
• Physical maps tell about the spatial
arrangement of physical “landmarks” such as
restriction sites.
 In restriction mapping cut the DNA in question with
2 or more restriction enzymes in separate reactions
 Measure the sizes of the resulting fragments
 Cut each with another restriction enzyme and
measure size of subfragments by gel electrophoresis
• Sizes permit location of some restriction sites
relative to others.
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5.6 Mapping and Quantifying Transcripts

 In the field of molecular biology mapping

(locating start and end) and quantifying (how
much transcript exists at a set time)
transcripts are common procedures.
 Often transcripts do not have a uniform
terminator, resulting in a continuum of
species smeared on a gel.
 Techniques that are specific for the
sequence of interest are important.

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 Northern Blots
 Northern blots detect RNA
 Example: You have cloned a cDNA
 Question: How actively is the
corresponding gene expressed in
different tissues?
 Answer: Find out using a Northern Blot
• Obtain RNA from different tissues
• Run RNA on agarose gel and blot to
membrane
• Hybridize to a labeled cDNA probe
Northern blot tells abundance of the
transcript
Quantify using densitometer
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 S1 Mapping
 Use S1 mapping to locate the ends of RNAs
and to determine the amount of a given RNA
in cells at a given time.
Label a ssDNA probe that can only hybridize to
transcript of interest
Probe must span where sequence starts or ends
After hybridization, treat with S1 nuclease
which degrades only ssDNA and RNA
Transcript protects part of the probe from
degradation
Size of protected area can be measured by gel
electrophoresis 5-30
S1 Mapping the 5’ End

5-31
S1 Mapping the 3’ End

5-32
 Summary
 A Northern blot is similar to a Southern blot
but is a method used for detection of RNA.
 In S1 mapping, a labeled DNA probe is used
to detect 5’- or 3’-end of a transcript.
 Amount of probe protected is proportional
to concentration of transcript, so S1 mapping
can be quantitative.
 RNase mapping is a variation on S1 mapping
that uses an RNA probe and RNase.

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 Run-Off Transcription
 A good assay to measure the
rate of in vitro transcription.
 DNA fragment containing
gene to transcribe is cut with
restriction enzyme in middle
of transcription region.
 Transcribe the truncated
fragment in vitro using labeled
nucleotides, as polymerase
reaches truncation it “runs
off” the end.
 Measure length of run-off
transcript compared to
location of restriction site at
3’-end of truncated gene. 5-34
Schematic of the G-Less Cassette Assay
 A variation of the run-off technique in which a stretch
of nucleotides lacking guanines is inserted into the
nontemplate strand just downstream of the promoter.
 Transcribe altered
template in vitro with CTP,
ATP and UTP one of which
is labeled, but no GTP.
 Transcription will stop
when the first G is
required resulting in an
aborted transcript of
predictable size.
 Separate transcripts on a
gel and measure
transcription activity with
autoradiography. 5-35
 Summary
 Run-off transcription is a means of checking
efficiency and accuracy of in vitro transcription.
 Gene is truncated in the middle and transcribed in
vitro in presence of labeled nucleotides
 Size of run-off transcript locates transcription start
site
 Amount of transcript reflects efficiency of
transcription
 In G-less cassette transcription, a promoter is
fused to dsDNA cassette lacking Gs in
nontemplate strand.
 Construct is transcribed in vitro in absence of GTP
 Transcription aborts at end of cassette for a
predictable size band on a gel
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 Reporter Gene Transcription
 Place a surrogate reporter gene under the
control of a specific promoter and measure
the accumulation of the product of this
reporter gene.
 The reporter genes are carefully chosen to
have products very convenient to assay
– lacZ produces -galactosidase which has a blue
cleavage product
– cat produces chloramphenicol acetyl transferase
(CAT) which inhibits bacterial growth (see figure
5.34)
– Luciferase produces a chemiluminescent compound
that emits light
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5.8 Assaying DNA-Protein Interactions

 Study of DNA-protein interactions is of

significant interest to molecular
biologists.
 Types of interactions often studied:
– Protein-DNA binding
– Which bases of DNA interact with a protein

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 Filter Binding
 Filter binding is used to measure DNA-
protein interaction, and based on the
fact that double-stranded DNA will not
bind by itself to a filter, but a protein-
DNA complex will.
– Double-stranded DNA can be labeled and
mixed with protein
– Assay protein-DNA binding by measuring
the amount of label retained on the filter

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 Nitrocellulose Filter-Binding Assay
 dsDNA is labeled and mixed with protein.
 Pour dsDNA through a nitrocellulose filter.
 Measure amount of radioactivity that passed
through filter and retained on filter.

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 Gel Mobility Shift
 DNA moves through a gel faster when it is
not bound to protein.
 Gel shift assays detect interaction between
protein and DNA by reduction of the
electrophoretic mobility of a small DNA
bound to a protein.

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 Footprinting

 Footprinting detects protein-DNA

interaction and will show where a target
lies on DNA and which bases are
involved in protein binding.
 Three methods are very popular:
DNase I footprinting
Dimethylsulfate footprinting
Hydroxyl radical footprinting

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 DNase I Footprinting
 Protein binding to DNA
covers the binding site and
protects from attack by
DNase I.
 End label DNA, 1 strand only
 Protein binds DNA
 Treat complex with DNase I
mild conditions for average
of 1 cut per molecule
 Remove protein from DNA,
separate strands and run on
a high-resolution
polyacrylamide gel

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 Summary
 Footprinting finds target DNA sequence or binding
site of a DNA-binding protein.
 DNase I footprinting binds protein to end-labeled
DNA target, then attacks DNA-protein complex
with DNase I.
 DNA fragments are electrophoresed with protein
binding site appearing as a gap in the pattern where
protein protected DNA from degradation.
 DMS, DNA methylating agent is used to attack the
DNA-protein complex.
 Hydroxyl radicals: copper- or iron-containing
organometallic complexes generate hydroxyl radicals
that break the DNA strands.
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 Chromatin Immunoprecipitation
(ChIP)

 ChIP is a method used to discover whether a

given protein is bound to a given gene in
chromatin: the DNA-protein complex that is
the natural state of the DNA in a living cell.
 ChIP uses an antibody to precipitate a
particular protein in complex with DNA, and
PCR to determine whether the protein binds
near a particular gene.

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Chromatin Immunoprecipitation
(ChIP)

5-46
 5.9 Assaying Protein-Protein
Interactions
 Immunoprecipitation uses an antibody that
will bind specifically to the protein of interest
and, using a low-speed centrifuge, will pull-
down any proteins associated with the protein
of interest.
 The yeast-two-hybrid assay is used to
demonstrate binding (even transient) between
two proteins.
 The yeast-two-hybrid assay can also be used
to fish for unknown proteins that interact
with a known protein.

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The Yeast-Two Hybrid Assay

5-48
 Student Expected Learning Outcomes
1. Discuss experiments used by molecular biologists to
separate nucleic acids and proteins.
2. Explain how gel electrophoresis, ion exchange
chromatography, and gel filtration can be used to
separate nucleic acids and proteins.
3. Understand and describe how labeled tracers are
used to detect small amounts of DNA, RNA and
proteins.
4. Explain the various assays used to detect protein-
DNA and protein-protein interactions, and know
when to apply them.

5-58